1. Field of the Invention
A method of identifying useful nucleic acid ligands with high affinity for target species is described. The target species may be an organism, a protein or other biopolymer or a small molecule. The nucleic acid ligands are characterized by a pre-defined primary and secondary structure which is retained in the final product.
2. Description of the Related Art
Nucleic acid constructs have been shown to have apparent affinities and selectivities that rival or exceed complexes with antibodies (Jayasena, S. D. (1999) Clin Chem, 45, 1628-1650; Gold, L. (1995) J Biol Chem, 270, 13581-13584). In addition, nucleic acids have been found that have high affinity and specificity for molecules that are too small to be immunogenic (Jenison, R. D., Gill, S. C., Pardi, A. and Polisky, B. (1994) Science, 263, 1425-1429). Antibody-based receptors cannot be created for most nerve gas agents and many common environmental contaminants. A sensor based on nucleic acid technology avoids many of the problems associated with antibody-based receptors and is applicable to both biological and chemical agents of a wide variety. The well-established methodology for discovery of high-affinity nucleic acid species used in these technologies is often referred to as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (U.S. Pat. Nos. 5,475,096 & 5,760,637; Tuerk, C. and Gold, L. (1990) Science, 249, 505-510); or in vitro selection (Ellington, A. D. and Szostak, J. W. (1990) Nature, 346, 818-822). This method uses iterative cycles of selection, amplification and cloning to discover target sequences known as “aptamers”.
SELEX is a process for discovering a DNA or RNA aptamer. This method begins with a solution of DNA molecules that are a mixture of 1013 to 1014 possible sequences that are a small and unknown subset of all possible sequences. These molecules have randomized, “variable” sequence regions that are usually 30-50 nucleotides (N30 or N50), but may be as large as 120 nucleotides in length or, in principle, larger. A starting pool with 1014 sequences contains only a tiny fraction of the diversity in such variable regions; for instance these pools contain only these fractions of all possible sequences: N30 (˜105), N50 (˜1016), N120 (˜1058), The variable region is flanked by fixed regions used in the amplification step. The target for selection of a high affinity sequence is mixed with this collection of sequences and the sequences that bind to the target are separated from those that do not bind as strongly (partitioning). A crucial washing step separates the bound and unbound species. The selected sequences are then amplified using nucleic acid enzymes. This process is repeated with more stringent requirements for affinity. Each cycle of selection and amplification enriches the sequence pool with fewer and fewer sequences. After repeating this process 9-15 times, the final high-affinity sequence pool is then cloned and each clone is sequenced. Examination of similarities in the resulting sequences may suggest a common tight-binding core sequence.
SELEX is very cumbersome, is prone to errors and is expensive to automate. The repetitive enzymatic/purification steps are cumbersome and time-consuming, often taking about 1 month to complete. Multiplexing requires expensive robotic equipment with frequent human interaction. The resulting sequences are generally larger than the minimal tight-binding sequences and sometimes very much larger than the minimal tight-binding sequence. The resulting aptamers tend to be large (50-100 nucleotides) and sometimes lack a defined secondary structure which limits their utility. This occurrence may be addressed by carving away the non-essential regions of a full-length aptamer to home in on the minimal tight binding aptamer “core”. This is accomplished by preparing constructs with residues removed from each end of the aptamer and assessing binding affinity. This leaves a core. Quite often, these core sequences (15-30 bases) that retain a high affinity for the target exist as unbranched stem-loops containing mismatches, internal loops, and apical hairpin loops.
This carving procedure was used to define core binding sequences in the aptamer for the HIV-1 nucleocapsid protein (Lochrie, M. A., Waugh, S., Pratt, D. G., Clever, J., Parslow, T. G. and Polisky, B. (1997) Nucleic Acids Res, 25, 2902-2910; Berglund, J. A., Charpentier, B. and Rosbash, M. (1997) Nucleic Acids Res, 25, 1042-1049) and many other proteins. It has also identified core sequences that bind tightly to small molecules, such as the anti-asthmatic drug, theophylline, and antibiotics such as tobramycin (Jiang, L. and Patel, D. J. (1998) Nat Struct Biol, 5, 769-774). Distinguishing the binding core by carving away the non-essential regions is a lengthy iterative process. It is also prone to errors. We have demonstrated this to be so for the NC-binding core sequences derived from aptamers (Lochrie, M. A., Waugh, S., Pratt, D. G., Clever, J., Parslow, T. G. and Polisky, B. (1997) Nucleic Acids Res, 25, 2902-2910; Berglund, J. A., Charpentier, B. and Rosbash, M. (1997) Nucleic Acids Res, 25, 1042-1049), which are all about twice as large as the minimal binding sequence and have led to aptamer cores that bind multiple NC proteins (Paoletti, A. C., McPike, M. P., Yule, R., Hudson, B. S. and Borer, P. N. submitted for publication). While the parent aptamer must contain a high affinity binding sequence, it must also present it in an appropriate secondary/tertiary structural context. The carving procedure, a necessary final step in converting the products of SELEX to usable form, can destroy the context and allow different modes of binding to operate.
A similar example emphasizes that aptamer cores are larger than minimal tight-binding sequences. An aptamer core sequence with high affinity for theophylline was determined by carving away non-essential residues (Jenison, R. D., Gill, S. C., Pardi, A. and Polisky, B. (1994) Science, 263, 1425-1429; Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, J. P. and Pardi, A. (1997) Nat Struct Biol, 4, 644-649). Anderson et. al. (Anderson, P. C. (2005) J. Am. Chem. Soc., 127 (15), 5290-5291, 2005) recently refined the core binding domain to a 13-mer hairpin loop structure with stem mismatches. The 13-mer displayed similar affinity and selectivity to the longer 33-mer aptamer discovered by SELEX (Jenison, R. D., Gill, S. C., Pardi, A. and Polisky, B. (1994) Science, 263, 1425-1429). This refinement relied on a 3D structure of the aptamer (Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, J. P. and Pardi, A. (1997) Nat Struct Biol, 4, 644-649), then performed molecular dynamics simulations after removing residues from the aptamer core that are likely to be non-essential for binding. The 13-mer preserved essential H-bonding and stacking characteristics of the 33-mer. It had a Kd˜10 μM and discriminated against caffeine by a factor of 40 (caffeine differs from theophylline by a single methyl group).
Embodiments of the present invention address the technical problems discussed above. Embodiments of the present invention differ from the SELEX method in that species with known primary and secondary structure are used. Therefore, candidate molecules have a defined secondary structure. The particular molecules selected from a library of candidate molecules has a defined secondary structure. Furthermore, there is no need to sequence the nucleic acid at any step in the process. Embodiments of the present invention use physical methods of affinity determination. Enzymatic amplification steps are not needed and there is no need to separate species on the basis of their affinity. Non-nucleic acid components may be incorporated. The practice of embodiments of the invention provides information on the affinity of species of known sequence which are not the strongest binding and results in a sequence that constitutes a minimized binding unit. There is no need to carve out a minimal tight binding core from a larger sequence. Constructs produced according to embodiments of the invention, in contrast to SELEX, may be readily incorporated into a biological switch.
Methods to rapidly discover nucleic acid oligomers that have high affinity and high specificity for protein and cellular targets are described. The resulting structures can easily be incorporated into bistable molecular sensors, such as OrthoSwitches™ (OrthoSystems, Inc.). The development of nucleic acid-based “capture” technologies represents an opportunity, currently unmet, in the entire area of sensors including those for air, food and water quality control, in medical diagnostics and in drug discovery.